Obesity-associated type-2 diabetes and its comorbidities, atherosclerosis and stroke, are all too common diseases in the aging population of our Veterans. These illnesses are invariably linked to aberrant lipid homeostasis as manifested by excessive synthesis and high circulating levels of very low-density lipoproteins and triglycerides. The long-term objective of our studies is to gain molecular insights into the mechanisms of hepatic lipid homeostasis and dyslipidemia and their cardiovascular complications. As a transcription factor that regulates key genes involved de novo lipid synthesis in the liver, sterol regulatory element binding protein-1c (SREBP-1c) is a central player in these processes. Nascent pSREBP-1c resides in the endoplasmic reticulum (ER) and must be transported to the Golgi where it undergoes regulated intra- membrane proteolysis (RIP) to release its transcriptionally active nuclear domain, nSREBP-1c that activates its own promoter. This feed-forward regulation SREBP-1c in the liver by insulin is a key mechanism of selective insulin resistance and hyperlipidemia and their clinical consequences. Based on compelling published data and our ongoing studies we hypothesize that phosphorylation via insulin-induced kinase PI3K-Akt/PKB, and its downstream kinases mTOR and p70S6K, regulate the RIP-mediated maturation, and trans-activation potency and stability of nSREBP-1c in the nucleus. Additionally, via crosstalk with insulin signaling kinases, MAP kinases also impinge on the feed-forward regulation of SREBP-1c in chronically obese rodents and man. With a goal to experimentally test this hypothesis, we have site-specifically mutated seventeen, AKT/mTOR/S6K and MAPK specific putative phosphorylation sites, selected on the basis of published observations and our theoretical analyses. Based on a rigorous assessment of the phenotypes of WT and mutated SREBP-1c in a McArdle hepatoma cell line and primary rat hepatocytes, we have selected FOUR sites [mTOR (S57 and S140), p70S6K (S956) and MAP kinase (S39)] for detailed examination of their role in the feed-forward regulation of SREBP-1c. The short-term goals of our proposal are (i) to identify sequence motifs of SREBP-1c involved in insulin-enhanced, phosphorylation- dependent regulation of RIP and trans-activation ability of nSREBP-1c in vitro and in vivo and (ii) to decipher how phosphorylation alters the molecular interactions of SREBP-1c during its ER to Golgi transit and proteolysis by S1P and S2P proteases, and (iii) to elucidate how phosphorylation alters the stability and transcriptional function of nSREBP-1c in the nucleus, in vitro and in vivo. To accomplish these goals, we will express wild type, and gain- or loss-of-phosphorylation mutants of tagged pSREBP-1c and nSREBP-1c proteins in rat hepatocytes in culture, and in livers of normal and obese and hyper-insulinemic MC4R knockout, rats, using our unique custom-designed adenovirus and plasmid vectors. We will compare the rates of RIP-mediated maturation of WT and mutated nSREBP-1c proteins and their trans-activation potential in the presence and absence of insulin. We will determine if/how phosphorylation alters the molecular interactions between full-length SREBP-1c and other molecules that are involved in ER-to-Golgi transit and proteolysis. Additionally, we will elucidate how phosphorylation alters the trans-activation potential of nSREBP-1c to activate its own promoter and the promoters of downstream target genes that regulate hepatic lipid synthesis. Our Specific Aims are designed to shed mechanistic light on phosphorylation-dependent actions of SREBP-1c on lipid homeostasis in vitro and in vivo. A successful completion of these studies has the potential to unravel novel therapeutic targets to treat chronic obesity and its pathological consequences for the cardiovascular system.